Measurement of cystic fibrosis transmembrane conductance regulator activity using fluorescence spectrophotometry

Abstract: Cystic fibrosis (CF) is a frequent autosomal recessive disease caused by mutations that impair the CF transmembrane conductance regulator (CFTR) protein function. CFTR is a chloride channel activated by cyclic AMP (cAMP) via protein kinase A (PKA) and ATP hydrolysis. We describe here a m...

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Autores principales: Valdivieso, Ángel Gabriel, Marín, María C., Clauzure, Mariángeles, Santa Coloma, Tomás Antonio
Formato: Artículo
Lenguaje:Inglés
Publicado: Elsevier 2022
Materias:
FIBROSIS QUÍSTICA
CANAL DE CLORURO
ESPECTROFOTOMETRÍA
REGULADOR DE CONDUCTANCIA TRANSMEMBRANA DE LA FIBROSIS QUÍSTICA
FLUORESCENCIA
Acceso en línea:https://repositorio.uca.edu.ar/handle/123456789/14595
Aporte de:
Repositorio Institucional de la Universidad Católica Argentina (UCA) de Universidad Católica Argentina
id I33-R139-123456789-14595
record_format dspace
spelling I33-R139-123456789-145952023-11-23T17:14:53Z Measurement of cystic fibrosis transmembrane conductance regulator activity using fluorescence spectrophotometry Valdivieso, Ángel Gabriel Marín, María C. Clauzure, Mariángeles Santa Coloma, Tomás Antonio FIBROSIS QUÍSTICA CANAL DE CLORURO ESPECTROFOTOMETRÍA REGULADOR DE CONDUCTANCIA TRANSMEMBRANA DE LA FIBROSIS QUÍSTICA FLUORESCENCIA Abstract: Cystic fibrosis (CF) is a frequent autosomal recessive disease caused by mutations that impair the CF transmembrane conductance regulator (CFTR) protein function. CFTR is a chloride channel activated by cyclic AMP (cAMP) via protein kinase A (PKA) and ATP hydrolysis. We describe here a method to measure CFTR activity in a monolayer of cultured cells using a fluorescence spectrophotometer and the chloridesensitive probe 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ). Modifying a slice holder, the spectrophotometer quartz cuvette was converted in a perfusion chamber, allowing measurement of CFTR activity in real time, in a monolayer of T84 colon carcinoma cells. The SPQ Stern–Volmer constant (KCl ) for chloride in water solution was 115.0 ± 2.8 M1 , whereas the intracellular KCl was 17.8 ± 0.8 M1 , for T84 cells. A functional analysis was performed by measuring CFTR activity in T84 cells. The CFTR transport inhibitors CFTR(inh)-172 (5 lM) and glibenclamide (100 lM) showed a significant reduction (P < 0.05) in CFTR activity. This simple method allows measuring CFTR activity in a very simple, reproducible, and sensitive way. 2022-08-02T13:36:07Z 2022-08-02T13:36:07Z 2011 Artículo Valdivieso, Á. G. Measurement of cystic fibrosis transmembrane conductance regulator activity using fluorescence spectrophotometry [en línea]. Analytical Biochemistry Bioquímica Analítica. 2011, 418 (2). doi: 10.1016/j.ab.2011.07.029. Disponible en: https://repositorio.uca.edu.ar/handle/123456789/14595 0003-2697 1096-0309 (online) https://repositorio.uca.edu.ar/handle/123456789/14595 10.1016/j.ab.2011.07.029 21864494 eng Acceso restringido http://creativecommons.org/licenses/by-nc-sa/4.0/ application/pdf Elsevier Analytical Biochemistry Bioquímica Analítica Vol. 418, No. 2, 2011
institution Universidad Católica Argentina
institution_str I-33
repository_str R-139
collection Repositorio Institucional de la Universidad Católica Argentina (UCA)
language Inglés
topic FIBROSIS QUÍSTICA
CANAL DE CLORURO
ESPECTROFOTOMETRÍA
REGULADOR DE CONDUCTANCIA TRANSMEMBRANA DE LA FIBROSIS QUÍSTICA
FLUORESCENCIA
spellingShingle FIBROSIS QUÍSTICA
CANAL DE CLORURO
ESPECTROFOTOMETRÍA
REGULADOR DE CONDUCTANCIA TRANSMEMBRANA DE LA FIBROSIS QUÍSTICA
FLUORESCENCIA
Valdivieso, Ángel Gabriel
Marín, María C.
Clauzure, Mariángeles
Santa Coloma, Tomás Antonio
Measurement of cystic fibrosis transmembrane conductance regulator activity using fluorescence spectrophotometry
topic_facet FIBROSIS QUÍSTICA
CANAL DE CLORURO
ESPECTROFOTOMETRÍA
REGULADOR DE CONDUCTANCIA TRANSMEMBRANA DE LA FIBROSIS QUÍSTICA
FLUORESCENCIA
description Abstract: Cystic fibrosis (CF) is a frequent autosomal recessive disease caused by mutations that impair the CF transmembrane conductance regulator (CFTR) protein function. CFTR is a chloride channel activated by cyclic AMP (cAMP) via protein kinase A (PKA) and ATP hydrolysis. We describe here a method to measure CFTR activity in a monolayer of cultured cells using a fluorescence spectrophotometer and the chloridesensitive probe 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ). Modifying a slice holder, the spectrophotometer quartz cuvette was converted in a perfusion chamber, allowing measurement of CFTR activity in real time, in a monolayer of T84 colon carcinoma cells. The SPQ Stern–Volmer constant (KCl ) for chloride in water solution was 115.0 ± 2.8 M1 , whereas the intracellular KCl was 17.8 ± 0.8 M1 , for T84 cells. A functional analysis was performed by measuring CFTR activity in T84 cells. The CFTR transport inhibitors CFTR(inh)-172 (5 lM) and glibenclamide (100 lM) showed a significant reduction (P < 0.05) in CFTR activity. This simple method allows measuring CFTR activity in a very simple, reproducible, and sensitive way.
format Artículo
author Valdivieso, Ángel Gabriel
Marín, María C.
Clauzure, Mariángeles
Santa Coloma, Tomás Antonio
author_facet Valdivieso, Ángel Gabriel
Marín, María C.
Clauzure, Mariángeles
Santa Coloma, Tomás Antonio
author_sort Valdivieso, Ángel Gabriel
title Measurement of cystic fibrosis transmembrane conductance regulator activity using fluorescence spectrophotometry
title_short Measurement of cystic fibrosis transmembrane conductance regulator activity using fluorescence spectrophotometry
title_full Measurement of cystic fibrosis transmembrane conductance regulator activity using fluorescence spectrophotometry
title_fullStr Measurement of cystic fibrosis transmembrane conductance regulator activity using fluorescence spectrophotometry
title_full_unstemmed Measurement of cystic fibrosis transmembrane conductance regulator activity using fluorescence spectrophotometry
title_sort measurement of cystic fibrosis transmembrane conductance regulator activity using fluorescence spectrophotometry
publisher Elsevier
publishDate 2022
url https://repositorio.uca.edu.ar/handle/123456789/14595
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AT clauzuremariangeles measurementofcysticfibrosistransmembraneconductanceregulatoractivityusingfluorescencespectrophotometry
AT santacolomatomasantonio measurementofcysticfibrosistransmembraneconductanceregulatoractivityusingfluorescencespectrophotometry
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